DESCRIPTION (provide by applicant): Identifying the fundamental mechanisms and components of protein trafficking in trypanosomes is critical for understanding the interaction of this parasite with its host. The development of systems for introducing and expressing heterologous genes in trypanosomes allows investigations at the molecular level of the factors mediating endocytic and exocytic processes. The recent identification of a lysozme membrane glycoprotein, p67, from the African trypanosome, T. brucei, and a homolog in the related South American trypanosome, T. cruzi, should by analogy to vertebrate lysosome proteins enable the identification of the cytosolic components required for cargo loading and vesicle formation. The innate targeting signals in p67 will be delineated and subsequently used to purify and clone the cytoplasmic constituents that mediate lysosome targeting of this membrane glycoprotein. Reporters will be constructed that are targeted to the lysosome by fusion with the cytoplasmic domain that mediates targeting. This domain will also be used as bait in both biochemical (affinity purification) and genetic (yeast two-hybrid analysis) approaches to isolate p67CD-binding protein. These cytosolic proteins can be expected to function in cargo loading and vesicle formation and thus be critical for secretory and endocytic protein trafficking in trypanosomes.